Despite being fundamental to vision science, the molecular basis of photopigments' spectral tuning remains poorly understood. Spectral shifts caused by substitutions at any opsin residue depend on genetic background, which means that designing an experiment to elucidate the functional consequences of opsin residue substitutions at each position is impossible. Evolution, on the other hand, has been experimenting on visual systems for millions of years and offers a unique opportunity to understand how spectral shifts occur. Animals live in very diverse light environments and some exhibit a spectacular diversity of colors. Studying how animal visual systems adapt to such variable conditions makes it possible to elucidate visual pigments spectral tuning in vertebrates, including humans (Yokoyama 1997; 2008). Unlike most mammals, birds have excellent color vision, and provide the best model system for uncovering diversity in the visual system of terrestrial vertebrates. However, vision in birds remains understudied. The proposed research aims to evaluate how the visual systems of closely related species of birds have adapted to different conditions and visual stimuli. Complete coding sequences for all cone opsin genes in 15 New World warblers indicate there is substantial amino acid variation in warbler opsins. Nearly 80% of the observed substitutions are in, or adjacent to the binding pocket, and therefore, have the potential to alter the opsins' spectral sensitivity. The functional consequences of mutations at each of these sites will be assessed by regenerating, expressing and measuring the spectral sensitivity of all the observed photopigment variants in vitro. After studying the molecular mechanisms of spectral tuning, we plan to evaluate selective forces that have shaped the evolution of photopigments in birds. Tetrahedral color spaces will be used to evaluate how the observed differences in the photopigments of warblers translate into differences in color perception and discrimination, and how they relate to plumage and habitat properties. This analysis will also test for a possible evolutionary relationship between each species' color vision and the evolved plumage characteristics. The proposed research will fill a significant gap in our understanding of photopigment spectral tuning, thereby contributing to our current understanding of color vision, including the molecular basis of human color vision deficiencies. PUBLIC HEALTH RELEVANCE: Studying how vision evolves in organisms such as birds, is an important route towards improved understanding of human vision and vision deficiencies. Here, natural experiments provided by evolution, give us the opportunity to study the mechanisms of spectral tuning in visual pigments.